Bottom Line:
DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis).Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed.We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

Affiliation: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, USA.

ABSTRACT

Unlabelled: Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.

Importance: The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of "repelling" the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and "traps" it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

fig1: BODIPY-labeled daptomycin (BDP-DAP) staining of representative E. faecalis S613 (DAP-susceptible) (a to e) and R712 (DAP-resistant) (f to j) cells at increasing concentrations of the fluorescent antibiotic. The images within each panel show bacterial cells captured by fluorescence (top image) and phase-contrast microscopy (bottom image) (bars, 1 µm). (a and b) In E. faecalis S613, binding of the antibiotic in a “peppering” pattern is observed only at subinhibitory concentrations of BDP-DAP. (c to e) At higher BDP-DAP concentrations, saturation of the surface is observed with staining of the division septum (white arrows). (f to j) In E. faecalis R712, the “peppering” staining pattern and binding of the fluorescent antibiotic is observed at all concentrations without visualization of septa. Additional images selected from different microscopic fields are shown in Fig. S2 in the supplemental material.

Mentions:
In order to understand the mechanism of enterococcal daptomycin (DAP) resistance, we used a previously described clinically derived pair of E. faecalis strains, a vancomycin-resistant DAP-susceptible (Daps) strain and a DAP-resistant (Dapr) strain recovered from the bloodstream of a patient before and after DAP therapy (S613 and R712, respectively [Table 1]) (12). We initially examined the binding of DAP to the cell membrane of the Daps clinical strain, S613, versus its Dapr variant, R712. We used boron-dipyrromethene (BODIPY)-labeled DAP (BDP-DAP), a fluorescent derivative of DAP that retains antimicrobial activity (MICs of 8 and 32 µg/ml for strain S613 and R712, respectively) (Table 1), to visualize DAP interactions with the cell membrane. Figure 1a and b show that at subinhibitory concentrations, multiple foci of BDP-DAP were bound to the S613 cell membrane and were distributed throughout the cell (“peppering” pattern) but sparing the division septum. At concentrations at and above the MIC, BDP-DAP decorated the E. faecalis S613 cell envelope in a homogeneous pattern, with a clear preference for the division septum (Fig. 1c to e). Indeed, ca. 85% of cells in a microscopic field displayed a linear fluorescence pattern at the septa (108 cells examined, BDP-DAP concentration of 16 µg/ml) (see Fig. S2 in the supplemental material). The binding pattern of BDP-DAP to Dapr strain R712 was similar to that of strain S613 at low concentrations (Fig. 1f and g). Moreover, we were able to observe consistent binding of BDP-DAP to E. faecalis R712 at all concentrations tested (Fig. 1f to j). However, at increasingly higher BDP-DAP concentrations, R712 continued to exhibit the “peppering” pattern. Notably, this pattern excluded the midpoints of the cells where the main septal structures are localized (Fig. 1h to j). Even at 16 and 32 µg/ml, only a small proportion of R712 cells exhibited septal staining with BDP-DAP compared to S613 cells (20% [n = 104] and 49% [n = 110], respectively; P < 0.0001) (Fig. S2). To further confirm that septal exclusion of BDP-DAP from septa is critical in the mechanism of resistance, we quantitated the fluorescence intensity in S613 and R712 cells treated with increasing concentrations of BDP-DAP and normalized to protein contents of the samples. No differences in fluorescent intensities between S613 and R712 cells were observed at any of the BDP-DAP concentrations tested (2 to 32 µg/ml) (all P > 0.05) (Fig. S3), suggesting that the total amount of bound antibiotic was similar in S613 and R712 cells. These data suggest that exclusion of DAP from the main septal structures and diversion of the antibiotic to other sites of the cell membrane mediate, at least in part, DAP resistance in E. faecalis R712.

fig1: BODIPY-labeled daptomycin (BDP-DAP) staining of representative E. faecalis S613 (DAP-susceptible) (a to e) and R712 (DAP-resistant) (f to j) cells at increasing concentrations of the fluorescent antibiotic. The images within each panel show bacterial cells captured by fluorescence (top image) and phase-contrast microscopy (bottom image) (bars, 1 µm). (a and b) In E. faecalis S613, binding of the antibiotic in a “peppering” pattern is observed only at subinhibitory concentrations of BDP-DAP. (c to e) At higher BDP-DAP concentrations, saturation of the surface is observed with staining of the division septum (white arrows). (f to j) In E. faecalis R712, the “peppering” staining pattern and binding of the fluorescent antibiotic is observed at all concentrations without visualization of septa. Additional images selected from different microscopic fields are shown in Fig. S2 in the supplemental material.

Mentions:
In order to understand the mechanism of enterococcal daptomycin (DAP) resistance, we used a previously described clinically derived pair of E. faecalis strains, a vancomycin-resistant DAP-susceptible (Daps) strain and a DAP-resistant (Dapr) strain recovered from the bloodstream of a patient before and after DAP therapy (S613 and R712, respectively [Table 1]) (12). We initially examined the binding of DAP to the cell membrane of the Daps clinical strain, S613, versus its Dapr variant, R712. We used boron-dipyrromethene (BODIPY)-labeled DAP (BDP-DAP), a fluorescent derivative of DAP that retains antimicrobial activity (MICs of 8 and 32 µg/ml for strain S613 and R712, respectively) (Table 1), to visualize DAP interactions with the cell membrane. Figure 1a and b show that at subinhibitory concentrations, multiple foci of BDP-DAP were bound to the S613 cell membrane and were distributed throughout the cell (“peppering” pattern) but sparing the division septum. At concentrations at and above the MIC, BDP-DAP decorated the E. faecalis S613 cell envelope in a homogeneous pattern, with a clear preference for the division septum (Fig. 1c to e). Indeed, ca. 85% of cells in a microscopic field displayed a linear fluorescence pattern at the septa (108 cells examined, BDP-DAP concentration of 16 µg/ml) (see Fig. S2 in the supplemental material). The binding pattern of BDP-DAP to Dapr strain R712 was similar to that of strain S613 at low concentrations (Fig. 1f and g). Moreover, we were able to observe consistent binding of BDP-DAP to E. faecalis R712 at all concentrations tested (Fig. 1f to j). However, at increasingly higher BDP-DAP concentrations, R712 continued to exhibit the “peppering” pattern. Notably, this pattern excluded the midpoints of the cells where the main septal structures are localized (Fig. 1h to j). Even at 16 and 32 µg/ml, only a small proportion of R712 cells exhibited septal staining with BDP-DAP compared to S613 cells (20% [n = 104] and 49% [n = 110], respectively; P < 0.0001) (Fig. S2). To further confirm that septal exclusion of BDP-DAP from septa is critical in the mechanism of resistance, we quantitated the fluorescence intensity in S613 and R712 cells treated with increasing concentrations of BDP-DAP and normalized to protein contents of the samples. No differences in fluorescent intensities between S613 and R712 cells were observed at any of the BDP-DAP concentrations tested (2 to 32 µg/ml) (all P > 0.05) (Fig. S3), suggesting that the total amount of bound antibiotic was similar in S613 and R712 cells. These data suggest that exclusion of DAP from the main septal structures and diversion of the antibiotic to other sites of the cell membrane mediate, at least in part, DAP resistance in E. faecalis R712.

Bottom Line:
DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis).Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed.We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

Affiliation:
Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, USA.

ABSTRACT

Unlabelled: Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.

Importance: The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of "repelling" the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and "traps" it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.